This disclosure relates generally to the field of electrophoretic separation of bio-agents and particles, and more particularly, to systems and devices for focusing the bio-agents into regions of relatively high concentrations.
Electrophoresis is a separation technique most often applied to the analysis of biological or other polymeric samples. It has frequent application to analysis of proteins and DNA fragment mixtures. The high resolution of electrophoresis has made it a key tool in the advancement of biotechnology. Variations of this methodology are used for DNA sequencing, isolating active biological factors associated with diseases such as cystic fibrosis, sickle-cell anemia, myelomas, and leukemia, and establishing immunological reactions between samples on the basis of individual compounds. Electrophoresis is an extremely effective analytical tool because it does not affect a molecule's structure, and it is highly sensitive to small differences in molecular charge and mass.
Particles can be manipulated by subjecting them to traveling electric fields. Such traveling fields are produced by applying appropriate voltages to microelectrode arrays of suitable design. Traveling electric fields are generated by applying voltages of suitable frequency and phases to the electrodes.
This technique of using traveling electric fields relates to an important method for separation and sorting of large particles and cells referred to as dielectrophoresis. Dielectrophoresis is defined as the movement of a polarizable particle in a non-uniform electric field. Essentially, the force arises from the interaction of the field non-uniformity with a field induced charge redistribution in the separated particle.
Particles are manipulated using non-uniform electric fields generated by various configurations of electrodes and electrode arrays. As a general biotechnological tool, dielectrophoresis is extremely powerful. From a measurement of the rate of movement of a particle the dielectric properties of the particle can be determined. More significantly, particles can be manipulated and positioned at will without physical contact, leading to new methods for separation technology.
A powerful extension of dielectrophoresis separation is traveling wave dielectrophoresis (TWD) in which variable electric fields are generated in a system of electrodes by applying time varying electric potential to consecutive electrodes. Such a method of Traveling Wave Field Migration was described by Parton et al. in U.S. Pat. No. 5,653,859, herein incorporated by reference. Although satisfactory, a need for improved strategies and methodologies remains. In addition, dielectrophoresis requires higher voltage (˜100 V), higher frequencies (˜10 MHZ), and finer electrode pitch (<10 um).
A microfluidic device for electrophoretic separation of biomolecules such as DNA and protein was described by Dunphy et al. in “Rapid Separation and Manipulation of DNA by a Ratcheting Electrophoresis Microchip (REM),” Proceedings of IMECE2002, Nov. 17-22, 2002, New Orleans, La., No. IMECE2002-33564, herein incorporated by reference. The device utilizes thousands of electrodes along the length of a microchannel. An electrical potential is applied across the electrodes and selectively varied to separate molecules within the microchannel into two groups using a ratcheting mechanism. This mechanism does not employ traveling waves. Although directed to the separation of biomolecules, this strategy is based upon micro device technology and is not readily compatible with conventional laboratory equipment. Accordingly, a need exists for a device and technique for utilizing electrostatic traveling waves for selectively concentrating bio-agents and particles, and particularly, for subsequent analysis.
Bio-agents dispersed either in aerosol form or in water are typically in such low concentrations that they are below the limit of detection (LOD) of even the most sensitive detection schemes. Yet, the ingestion of even a single bacterium may lead to fatal consequences. Aerosol collection schemes typically sample large volumes of air at very high rates (up to 150 kl/min), and use a cyclone or a virtual impactor to collect particles of size in the threat range and capture them in a wet sample of 5-10 ml volume. This hydrosol is then used as the test sample for agent detection.
Contaminants in water are typically treated by several filtration steps to recover the sample for agent testing. After initial pre-filtration to remove large, vegetative matter, the sample is further concentrated by two to three orders of magnitude using ultra-filtration. This method of tangential flow filtration (TFF) is laborious as it requires multiple sequential steps of TFF; each step with a filter of lower molecular weight cut-off, and recycling of the retentate. The supernatant at the end is the 50 ml volume to be presented to the detector. Regardless of whether the sample is derived from aerosol or water collection, it would be useful to have a device to further concentrate the sample prior to detection, preferably by several orders of magnitude and within a smaller volume in the 50-100 μl range.